Genetic engineering has provided a method to isolate, selectively amplify, and express genes encoding desirable traits. These genes are often obtained from one organism and transformed into another organism so that expression of the gene can be manipulated and maximized. The transfer of genes from one organism to another can be used to produce large quantities of the gene product as well as to provide the transformed organism with improved characteristics or traits. Heterologous genes encoding desirable traits can be introduced into a wide variety of prokaryotic and eukaryotic hosts. For example, advantageous genes encoding herbicide resistance from bacteria can be incorporated into a plant""s genome. The bacterial gene can then be expressed in the plant cell to confer on the plant cell resistance to the herbicide.
In order for the newly inserted gene to be expressed in a eukaryotic or prokaryotic host cell, proper regulatory sequences must be present and in the proper location with respect to the coding sequence. These regulatory sequences include a promoter region and a 3xe2x80x2 nontranslated regulatory region. The promoter is a region of DNA sequences located upstream from the coding sequence of the gene. The function of a promoter sequence is to allow access and position of the transcription enzyme RNA polymerase in the vicinity of the transcription initiation site. The promoter DNA sequence can contain regulatory sequences that influence the rate and timing of the transcription of the gene. For example, insertion of a 21 base pair sequence into the 35S cauliflower mosaic virus (CaMV) promoter results in a tissue-specific expression in roots (Lam et al., PNAS, 86:7890 (1989)). Other sequences, like the TATA box and the CCAAT box in eukaryotic promoters, are known to influence the rate and level of gene transcription.
Certain promoters are known to be strong promoters. These promoters direct transcription at higher levels than other types of promoters and are capable of directing expression in other types of cells. One of the promoters that is a strong promoter in some types of plant cells is the 35S cauliflower mosaic virus (CaMV) promoter. However, the 35S CaMV promoter expression in plants can be variable and is especially so in monocotyledonous plants. Thus, strong promoters in one system often are not capable of providing for gene expression in a wide variety of both prokaryotic and eukaryotic host cells.
Chlorella viruses are a large group of recently identified viruses that infect certain eukaryotic green algae Chlorella. Chlorella viruses can be produced in large quantities and can be assayed by plaque formation. Chlorella viruses are large (150 to 190 nm) polyhedral plaque forming viruses containing greater than 300 kilobases of linear double-stranded DNA. The viruses are placed into 16 classes on the basis of plaque size antibody reactivity and the nature and abundance of methylated bases in their genomic DNA.
The Chlorella viruses have several unique features. The viruses have enough DNA sequence to encode 200 to 300 proteins. It is known that each virus contains and encodes 50 structural genes. The viruses also encode several DNA methyltransferase genes, DNA restriction endonuclease genes, and DNA polymerase genes. The DNA methyltransferase genes and the restriction endonuclease genes have been studied as a unique DNA restriction-modification system. Because the Chlorella viruses can be grown to large quantities and have several unique features, they are good candidates for the isolation of factors important in gene regulation.
Thus, there is a need for identifying and isolating strong promoters that are capable of expressing heterologous genes in a wide variety of cell types. There is also a need to identify and isolate strong promoters that can function in monocotyledonous plants, like wheat or rice. There is also a need to identify, isolate and characterize the promoters of the Chlorella virus genes including the DNA methyltransferase genes.
There is also a need for promoters of different strengths. For example different strength promoters are needed to express proteins at different levels which may otherwise be toxic to the organism at higher concentrations. Also needed are promoters which express efficiently in both prokaryotic and eukaryotic organisms, thus allowing for easy manipulation of a gene of interest. Also, there is a need for promoters which express efficiently in both prokaryotic and eukaryotic organisms, since most of the commonly used promoters do not work efficiently in monocots and dicots, thus making foreign gene introduction difficult.
The invention is directed to novel promoters or mutants thereof from Chlorella virus DNA methyltransferase genes. These novel promoters are operably linked to a first DNA sequence encoding a gene that is different from the Chlorella virus gene to form an expression cassette. To be functional in eukaryotic cells, an expression cassette typically includes a 3xe2x80x2 nontranslated regulatory DNA sequence functional in eukaryotic cells and operably linked to the first DNA sequence. The preferred Chlorella virus DNA methyltransferase promoters in the expression cassette provide for a high level of constitutive gene expression in prokaryotic and eukaryotic cell hosts.
An expression cassette of the invention can further comprise a second DNA sequence encoding a different gene from the first DNA sequence. The second DNA sequence is linked to the first DNA sequence and under the control of the Chlorella virus promoter. The second DNA sequence preferably encodes a reporter gene or a selectable marker gene.
An expression cassette of the invention is introduced into prokaryotic and eukaryotic cells, preferably in a plasmid vector. Plasmid vectors including an expression cassette of the invention are used to stably transform prokaryotic cells. The preferred transformed prokaryotic species include E. coli, phytopathogenic members of the genera Pseudomonas and Erwinia, plant associated members of the genus Xanthomonas, and members of the genus Agrobacterium. Stably transformed prokaryotic cells are selected and are capable of transmitting an expression cassette to progeny cells. The transformed progeny cells express the genes encoded by the first and/or second DNA sequence under the control of the Chlorella virus promoters.
Eukaryotic cells, preferably plant cells, can be transformed with an expression cassette, typically in a binary Ti vector. Transformed plant cells can transiently express the genes from the first and/or second DNA sequence. Transformed plant cells exhibiting transient gene expression, preferably monocotyledonous plant cells, can be converted to stably transformed or transgenic plants. Transformed plant cells are incubated in the presence of callus induction medium and a selective agent. Transformed calli can then be used to generate the transformed or transgenic plants. The transgenic plants can be grown and selfed or crossed to produce transgenic progeny plants and seeds. The preferred transgenic plant of the invention is a monocotyledonous plant having a Chlorella virus promoter that provides for a high level of constitutive gene expression.
The invention also provides a method for screening other Chlorella virus genes for promoters that can function to express a heterologous gene in prokaryotic and/or eukaryotic hosts. Chlorella virus genes with their 5xe2x80x2 flanking DNA sequences are isolated, sequenced, and the coding region of the gene identified. Once the Chlorella virus gene with its 5xe2x80x2 flanking sequence has been identified, a method of the invention involves isolating a DNA fragment including about 50 to 2000 nucleotide base pairs of the DNA sequence upstream from the coding sequence. An expression cassette is formed by combining the DNA fragment with a reporter gene, like chloramphenicol acetyltransferase. An expression cassette is used to transform prokaryotic and/or eukaryotic hosts and expression of the reporter gene is detected. Promoter sequences providing for a high level of gene expression in eukaryotic and/or prokaryotic hosts can be identified.